All references mentioned hereinbelow are listed at the end of this description and are herein incorporated by reference in their entirety.
Tuberous sclerosis (TSC) is an autosomal dominant disorder, classified as a phakomatosis (van der Hoeve, 1933) and characterised by the widespread development of growths, usually described as hamartomata, in many tissues and organs. The unpredictable distribution of these lesions, particularly within the brain, eyes, skin, kidneys, heart, lungs and skeleton results in a wide variety of signs, symptoms and complications (Gomez, 1988). Although most frequently diagnosed as a result of neurological or dermatological manifestations, renal disease was found to be the leading cause of mortality (11/40 deaths) in the largest series of TSC deaths reported so far. Renal complications including haemorrhage, hypertension and end stage renal disease (ESRD) are associated with the development of cysts and hamartomatous growths (angiomyolipomata) in the kidneys. Angiomyolipomata probably arise due to coexistent inactivating constitutional and somatic mutations, consistent with the TSC genes functioning as tumour or growth suppressor genes (Green et al (1994) and Green et al (in press)). Therefore the frequency of diagnosed cases is likely to under-represent true prevalence which may be as high as 1 in 5,800 (Osborne et al., 1991). The pathogenesis of TSC is poorly understood and efforts to establish the primary underlying defect have focused on positional cloning of the causative gene(s).
Linkage studies have established locus heterogeneity (Sampson et al., 1989a & 1992, Haines et al., 1991a&b, Janssen et al., 1991, Povey et al.,1991, Northrup et al., 1992) with disease determining genes on chromosomes 9 (Fryer et al., 1987) and 16 (Kandt et al., 1992) leading to apparently indistinguishable phenotypes. In most, if not all, affected multigeneration families the disease can be accounted for by the gene at one or other of these loci (Kwiatkowski et al., 1993). The Genome Database Nomenclature Committee recently agreed that the loci on chromosomes 9 and 16 should be termed TSC1 and TSC2 respectively. Analysis of meiotic recombination events in TSC families has refined the positions of TSC1 and TSC2 to small regions in the telomeric chromosomal bands 9q34.3 and 16p13.3. The candidate region at 16p13.3 extends between the markers MS205.2 (D16S309) and 16AC2.5 (D16S291) (Kwiatkowski et al., 1993), representing an estimated 1.5 megabases of DNA.
Loss of heterozygosity for alleles at 16p has been observed in hamartomata from TSC patients (Green and Yates, 1993; Smith et al., 1993), indicating that a second somatic mutation may be required to produce the TSC phenotype at a cellular level. This observation is consistent with the chromosome 16 TSC gene acting as a tumour suppressor, a feature shared by genes causing the other phakomatoses, neurofibromatosis type 1 (NF1) (Legius et al., 1993) and type 2 (NF2) (Trofatter et al., 1993), and von Hippel-Lindau disease (VHL) (Latif et al., 1993). If a two-hit mechanism, as proposed by Knudson (1971), does apply to TSC, then inactivating constitutional mutations would be anticipated. TSC has not been noted in individuals with the chromosome 16 .alpha.-thalassaemia/mental retardation syndrome (ATR-16) and terminal deletions of 16p which extend into the distal part of the candidate region (Wilkie et al., 1990). The inventors of the present invention therefore investigated the proximal part of the candidate region for deletions.
Some 60% of TSC cases appear to represent new mutations (Sampson et al., 1989b) and the inventors reasoned that a proportion of these might be large deletions. Such deletions, detectable by pulsed field gel electrophoresis (PFGE), would greatly facilitate identification of the gene, as has been demonstrated in NF1, NF2 and VHL (Viskochil et al., 1990; Trofatter et al., 1993; Latif et al., 1993). The inventors have now identified 5 TSC associated constitutional interstitial deletions of between 30 and 75 kb in the proximal part of the candidate region. These have been mapped to a 120kb segment from which the inventors have identified a number of genes, one of which was disrupted by all the deletions. Mutation analysis and expression studies provide strong evidence that this gene, which we term TSC2, is the chromosome 16 tuberous sclerosis determining gene.